Tag: protein

Catching light-activated proteins in action

Catching light-activated proteins in action

Light is an important feature of the natural world. Many organisms have developed sophisticated systems to detect light and then convey signals to sensory systems that respond. This can be achieved through coupled systems that contain both a light-sensing chromophore and a protein that passes on the information via protein conformational changes to other domains or proteins in the system.

However, these reactions work on very fast timescales and not much is known about the structural intermediates that are involved. This information is important for understanding how these systems work and could be useful for applications such as the design of light-activated cellular sensors for research or medical treatments.

In a recent publication, a collaborative team from the Korean Advanced Institute of Science and Technology (KAIST), the Korean Center for Advanced Reaction Dynamics, and the University of Chicago reported on their findings from work conducted at the University of Chicago’s BioCARS 14-ID-B beamline at the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory. Their results provide new structural and mechanistic insights to further illuminate this process.

The research focused on a light-sensing protein from the common oat plant, Avena sativa, called AsLOV2, a member of a superfamily of light-activated proteins that contain the light-oxygen-voltage (LOV) domain. These LOV domain-containing proteins detect blue light in the visible spectrum and have a conserved structure composed of five β sheets and four α helices. When blue light activates the chromophore, a covalent bond is formed between the light-sensing molecule and a cysteine amino acid on the protein. This is hypothesized to lead to protein dimerization and other conformational changes that transmit the light signal downstream.

The team used time-resolved X-ray liquidography (TRXL), a sensitive technique that can detect global conformational changes in solution on a millisecond to microsecond timescale, to analyze the light-activated transition of AsLOV2.

The structure of interest for the work was a piece of the full-length AsLOV2 protein that contained the LOV domain and two helices, A’α and Jα, that are known to be involved in the light-induced dimerization of the protein and downstream signaling. The team used a mutant–type of the protein (I427V) that has a faster recovery rate than the wild–type (WT) protein, facilitating some of the measurements. Kinetic evaluation of the TRXL data showed that light-induced transition of AsLOV2 includes ground (G), first intermediate (I1), second intermediate (I2), and final photoproduct (P) states with associated time constants (WT: 682 microseconds [μs] and 10.6 milliseconds [ms], and I427V: 130 μs and 3.4 ms).

Read more on APS website

A powerful tool for nanoparticles analysis in complex biological media

A powerful tool for nanoparticles analysis in complex biological media

An article published by CNPEM researchers was featured on the Nano Letters scientific journal’s cover and explores how the X-ray Photon Correlation Spectroscopy (XPCS) technique can distinguish protein corona formation from nanoparticle aggregation in complex biological media.

The innovative work, carried out at Sirius, expands analysis capacity in nanomedicine and highlights the XPCS potential to characterize nanoparticle interactions in biological environments in real time, providing a valuable resource for nanobiotechnology research and new biomedical materials development. 

The innovative nanoparticles applications in biomedicine

Nanoparticles are tiny structures, with dimensions generally between 1 and 100 nanometers. Due to its size, they can interact with cells, proteins and molecules in a highly precise way, which allows driven delivery of medicines and therapeutic agents. This allows, for example, for cancer treatments to be more effective, by releasing drugs directly into tumor cells, minimizing side effects on healthy tissues.

Furthermore, nanoparticles can be designed for responding to specific stimuli, such as pH, temperature or biological signs, allowing a controlled release of medicines only when necessary.

In the diagnosis area, nanoparticles offer new ways ​​to prematurely detect diseases. They can be linked to specific biomarkers that bind to molecular targets, making it easier to identify cancerous cells or the presence of viruses and bacteria, for example. 

The interaction between nanoparticles and proteins in biological systems

These applications, however, are conditioned to a predictable behavior of these nanoparticles in complex biological systems. In some cases, by coming into contact with biological fluids, such as blood, a protein coating can be formed around nanoparticles, a phenomenon known in biomedicine by the English term “protein corona”. 

This happens because nanoparticles attract proteins present in the biological environment, forming a “corona” or “crown” around its surface. The formation of this protein corona strongly influences how do nanoparticles interact with cells and tissues in the organism, which can affect its efficacy and safety in medical applications, such as drug therapies, diagnostics, and vaccine development. 

For these reasons, studying the protein corona formation and characteristics is crucial for the development of nanoparticles that are safe and effective for biomedical use. 

Read more on LNLS website

Image: Schematic representation of a functionalized SiO2 nanoparticle

Congratulations to the Nobel Prize winners in chemistry

Congratulations to the Nobel Prize winners in chemistry

The researchers at the world’s largest free-electron laser, the European XFEL, are delighted that Demis Hassabis, John M. Jumper and David Baker have been awarded the Nobel Prize in Chemistry. The decoding of protein structures is an important field of research for X-ray lasers such as the European XFEL.

David Baker has been an active user of the European XFEL since 2022. His team has actively participated in single-molecule imaging experiments at the Small Quantum Systems (SQS) and SPB/SFX instrument.

There, they recorded diffraction patterns of computationally designed proteins and single molecules for the first time.

“We are excited that David Baker has received the Nobel Prize for his ground-breaking work in the computer-aided design of de novo proteins”, says Thomas Feurer, Chairman of the Management Board of European XFEL. “We look forward to collaborating on upcoming experiments where he plans to explore the ultra-fast dynamics and behaviour of these innovative proteins with us.”

Read more on European XFEL website

Image: David Baker, Demis Hassabis and John Jumper. Ill. Niklas Elmehed

Credit: Nobel Prize Outreach

Newly discovered protein stops DNA damage

Newly discovered protein stops DNA damage

Researchers from Western University have discovered a protein that has the never-before-seen ability to stop DNA damage in its tracks. The finding could provide the foundation for developing everything from vaccines against cancer, to crops that can withstand the increasingly harsh growing conditions brought on by climate change.

The researchers found the protein – called DdrC (for DNA Damage Repair Protein C) — in a fairly common bacterium called Deinococcus radiodurans (D. radiodurans), which has the decidedly uncommon ability to survive conditions that damage DNA – for example, 5,000 to 10,000 times the radiation that would kill a regular human cell. Lead researcher Robert Szabla says Deinococcus also excels in repairing DNA that has already been damaged.“It’s as if you had a player in the NFL who plays every game without a helmet or pads,” says Szabla, a grad student in Western’s Department of Biochemistry. “He’d end up with a concussion and multiple broken bones every single game, but then miraculously make a full recovery overnight in time for practice the next day.” He and his colleagues discovered that DdrC is a key player in this repair process.

Read more on CLS website

Mechanistic Insight into a Viral-Factory Component

Mechanistic Insight into a Viral-Factory Component

“Viral factories” are areas in virus-infected host cells where the tools and materials necessary for viral replication are concentrated. In this study, researchers sought to learn more about an important component of some viral factories, a protein called σNS. This protein takes part in the replication of reoviruses, which are generally nonpathogenic and can be used as an oncolytic agent to target cancer cells. Despite its importance, the underlying mechanics of σNS have remained unclear.

A collaborative team led by B.V.V. Prasad at Baylor College of Medicine and Terence Dermody at the University of Pittsburgh conducted protein crystallography studies at Beamline 5.0.1 of the Advanced Light Source. They looked at a mutant version of σNS, σNS-R6A, which forms dimers rather than the longer chains (oligomers) of the unmutated protein, which resists crystallization.

The team discovered that σNS-R6A dimers interact by inserting protruding arms into a pocket of its neighbor, forming a helical assembly. The interior of the helical assembly is positively charged, making it suitable for binding RNA.

Bile acids were found to disrupt σNS assembly by binding the same pocket. “This was a serendipitous discovery,” said Prasad. “First author Boyang Zhao, a graduate student at the time, had set up crystallization trials with additives that included bile acid salts. When the crystal structure was determined, he saw bile acid moiety in the structure.”

Read more on ALS website

Image: Interacting dimers are shown in pink and blue, with the two monomeric subunits in the pink dimer labeled A and A’. The N-terminal arms (in red frames) project in opposite directions (red arrows) to chain-link the dimers to form a helical assembly.

Research on the structure of human cold receptor TRPM8

Research on the structure of human cold receptor TRPM8

Researchers from the Laboratory of Protein Structure at the International Institute of Molecular and Cell Biology in Warsaw, led by Prof. Marcin Nowotny, used the KRIOS cryoelectron microscope located at the SOLARIS National Synchrotron Radiation Centre to study the human TRPM8 protein.

The structure they obtained will enable a better understanding of the binding mechanism of small-molecule compounds affecting the activity of this ion channel. It will facilitate the design of new small-molecule compounds that can be used as therapeutics to treat numerous diseases associated with TRPM8 protein, such as neuropathic pain, irritable bowel syndrome, oropharyngeal dysphagia, chronic cough, and hypertension. As an example, in collaboration with scientists from Italy led by Dr. Carmine Talarico of Dompé Farmaceutici SpA, they have performed modeling of the binding of icilin, a small-molecule compound showing 200 times stronger TRPM8 channel activation than menthol. 

Read more on SOLARIS website

Image:  Structure of human cold receptor TRPM8

Credit: Mariusz Czarnocki-Cieciura

Stanford study shows how modifying enzymes’ electric fields boosts their speed

Stanford study shows how modifying enzymes’ electric fields boosts their speed

A seemingly subtle swap of metals—substituting a zinc ion with a cobalt ion—and a mutation ramps up the overall electric field strength at the active site of an enzyme, Stanford scientists find. The result is a predictably modified enzyme that works an astonishing 50 times faster than its unmodified analog.

Stanford researchers have demonstrated a way to dramatically speed up the reaction rate of an enzyme, a finding that could pave the way to designing ultra-fast synthetic enzymes for a range of industrial and medical uses.

Honed over billions of years of evolution, biological enzymes are marvels of chemistry. These specialized proteins serve as catalysts for accelerating chemical reactions essential to life as well as processes used in the food, pharmaceutical, and cosmetic industries.    

Ever since enzymes’ discovery nearly two centuries ago, scientists have sought ways to make them even faster. Most fabricated enzymes, though, have failed to match the lofty efficiency standards of nature-made varieties. And even where some successes have been realized through directed evolution, a protein engineering method that mirrors nature’s trial-and-error approach, these successes so far have been by chance, not because of a deeper understanding of how enzymes work or could be modified to work more swiftly.

Now, in a new study, researchers at Stanford’s School of Humanities and Sciences and SLAC National Accelerator Laboratory have debuted a modified enzyme that works an astonishing 50 times faster than its unmodified analog. The findings derive from pioneering research at the university regarding electric fields generated at “active sites,” the pocketlike places where revved up chemical reactions occur. Based on this concept, the researchers tweaked the chemistry of the active site, boosting its electric field strength and specificity to deliver the zippy results.  

Read more on Stanford University website

Image: X-ray crystallography was used to investigate and compare the 3D crystal structures of the unmodified enzyme containing an ion of zinc (Zn) (pictured left) and the modified enzyme with a cobalt (Co) ion in place of zinc (pictured right).

Newly identified protein could help fight cancer

Newly identified protein could help fight cancer

Researchers from the University of British Columbia (UBC) have identified a new protein that helps an oral bacterium thrive in other locations around the body. The discovery could eventually lead to the development of new drugs that specifically target the protein.

“This bacterium is common in the mouths of humans and generally doesn’t cause disease in that location. However, it can travel through the bloodstream to other areas of the body, which leads to some pretty big health concerns,” says Dr. Kirsten Wolthers, Associate Professor of Biochemistry and Microbiology at UBC’s Okanagan Campus.

Most notably, this bacteria is prevalent in the tumors of colorectal cancer patients. The presence of the bacteria can contribute to tumor growth, spread of cancer to other sites in the body, and resistance to chemotherapy.

With the help of the CMCF beamline at the Canadian Light Source (CLS), located at the University of Saskatchewan, Wolthers and her colleagues determined that the new protein they identified enables the bacteria to take essential nutrients, such as iron, from our blood cells.

Read more on the CLS website

Image: Alexis Gauvin, inspecting a protein sample for particulate matter, using the glove box. Gauvin is a biochemistry student and a member of Dr. Kirsten Wolthers’s research group in the Department of Chemistry, University of British Columbia (Okanagan Campus).

Picking up good vibrations – of proteins – at CHESS

Picking up good vibrations – of proteins – at CHESS

A new method for analyzing protein crystals – developed by Cornell researchers and given a funky two-part name – could open up applications for new drug discovery and other areas of biotechnology and biochemistry.

The development, outlined in a paper published March 3 in Nature Communications, provides researchers with the tools to interpret the once-discarded data from X-ray crystallography experiments – an essential method used to study the structures of proteins. This work, which builds on a study released in 2020, could lead to a better understanding of a protein’s movement, structure and overall function.

Protein crystallography produces bright spots, known as Bragg peaks, from the crystals, providing high-resolution information about the shape and structure of a protein. This process also captures blurry images – patterns and clouds related to the movement and vibrations of the proteins – hidden in the background of the Bragg peaks.

These background images are typically discarded, with priority given to the bright Bragg peak imagery that is more easily analyzed.

“We know that this pattern is related to the motion of the atoms of the protein, but we haven’t been able to use that information,” said lead author Steve Meisburger, Ph.D. ’14, a former postdoctoral researcher in the lab of Nozomi Ando, M.S. ’04, Ph.D. ’09, associate professor of chemistry and chemical biology in the College of Arts and Sciences. “The information is there, but we didn’t know how to use it.  Now we do.”

Meisburger worked closely with Ando to develop the robust workflow to decode the weak background signals from crystallography experiments called diffuse scattering. This allows researchers to analyze the total scattering from crystals, which depends on both the protein’s structure and the subtle blur of its movements.

Their two-part method – which the team dubbed GOODVIBES and DISCOBALL – simultaneously provides a high-resolution structure of the protein and information on its correlated atomic movements.

GOODVIBES analyzes the X-ray data by separating the movements – subtle vibrations – of the protein from other proteins that might be moving around it. DISCOBALL independently validates these movements for certain proteins directly from the data, allowing researchers to trust the results from GOODVIBES and understand what the protein might be doing.

Read more on CHESS website

Image: Meisburger, Case, & Ando (2020) Nat Commun 11, 1271

Sirius helps reveal previously unknown process of maturation for key protein in SARS-CoV-2 replication

Sirius helps reveal previously unknown process of maturation for key protein in SARS-CoV-2 replication

Researchers at USP in São Carlos combined cutting-edge technologies and demonstrated that a molecule targeted by medications behaves differently than previously theorized.

A group of researchers from the University of São Paulo in São Carlos has just presented their findings from research indicating a new understanding of the maturation process and how inhibitors act upon the Mpro protein, an essential component in the life cycle of the Sars-CoV-2 virus and the target of various efforts to develop medications to treat Covid-19. Their results appear in an article entitled “An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition,” published in the journal Nature Communications (https://doi.org/10.1038/s41467-023-37035-5).

Mpro is an abbreviation for main protease, because of its importance to the virus. Today, two medications are available which interact with this molecule to treat covid-19. Still, some of the processes in this protein’s activity are not yet entirely understood, and this was the object of the study undertaken at Sirius.

As part of the role it plays in the life cycle of the Sars-CoV-2 virus, Mpro undergoes a series of modifications until it reaches its final form. Part of this process had already been described by the group from São Carlos, directed by Professor Glaucius Oliva.

André Godoy, who led the group, was one of the first external users of Sirius, the cutting- synchrotron light source planned and built by the Brazilian Center for Research in Energy and Materials (CNPEM), an organization overseen by the Ministry of Science, Technology and Innovation (MCTI).

In September 2020 he brought approximately 200 crystals containing proteins from the Sars-CoV-2 virus for analysis in the Manacá beamline, which was developed for experiments involving X-ray diffraction crystallography. “The Manacá beamline was the first research station to open at Sirius, as the result of a task-force effort at the CNPEM to support research exploring molecular mechanisms related to covid-19. This is one of the publications that resulted from this effort,” explains Harry Westfahl, Director of the Brazilian Synchrotron Light National Laboratory (LNLS).

Read more on the LNLS website

Image: Cryomicroscopy map of the Mpro dimer interacting with the N-terminal. Image obtained from analyses conducted at Diamond and Sirius by the USP São Carlos group

How vision begins

How vision begins

Researchers at the Paul Scherrer Institute PSI have deciphered the molecular processes that first occur in the eye when light hits the retina. The processes – which take only a fraction of a trillionth of a second – are essential for human sight. The study has now been published in the scientific journal Nature.

It only involves a microscopic change of a protein in our retina, and this change occurs within an incredibly small time frame: it is the very first step in our light perception and ability to see. It is also the only light-dependent step. PSI researchers have established exactly what happens after the first trillionth of a second in the process of visual perception, with the help of the SwissFEL X-ray free-electron laser of the PSI.

At the heart of the action is our light receptor, the protein rhodopsin. In the human eye it is produced by sensory cells, the rod cells, which specialise in the perception of light. Fixed in the middle of the rhodopsin is a small kinked molecule: retinal, a derivative of vitamin A. When light hits the protein, retinal absorbs part of the energy. With lightning speed, it then changes its three-dimensional form so the switch in the eye is changed from “off” to “on”. This triggers a cascade of reactions whose overall effect is the perception of a flash of light.

Read more on the PSI website

Image: PSI researcher Valérie Panneels purifies the red protein rhodopsin in order to examine it later at the SwissFEL X-ray free-electron laser

Credit:  Scanderbeg Sauer Photography

Battling biofilm to prevent dangerous lung infections

Battling biofilm to prevent dangerous lung infections

Researchers from the University of Toronto (U of T) and The Hospital for Sick Children have identified a promising therapeutic target to help treat lung infections in cystic fibrosis (CF) patients.

“Individuals with cystic fibrosis have an impairment in their lungs where they have a hard time clearing out the mucus that accumulates within the lungs,” says Andreea Gheorghita, PhD candidate in the Department of Biochemistry at U of T.

Pseudomonas aeruginosa is a bacterium that causes opportunistic infections in individuals with weakened immune systems or other health concerns. For individuals with CF, repeated Pseudomonas infections often lead to long hospital stays and severe lung damage.

“Because of the impaired ability to clear mucus in the airways, these lung infections can become very persistent and prolonged, which eventually leads to lung tissue damage, loss of lung function, and eventually can cause patient mortality,” says Gheorghita.

Using the CMCF beamline at the Canadian Light Source (CLS) at the University of Saskatchewan (USask), the team has been able to visualize the interaction between two important proteins that are key players in Pseudomonas’s ability to make biofilm. This sticky secretion allows the bacterium to attach to the lungs and makes it difficult for antibiotics and the patient’s immune system to fight the infection.

Read more on the  Canadian Light Source website

Insights into coronavirus proteins using SAXS

Insights into coronavirus proteins using SAXS

A collaboration led by researchers from the European Molecular Biology Laboratory (EMBL) used small angle X-ray scattering (SAXS) at the European XFEL and obtained interesting data on samples containing coronavirus spike proteins including proteins of the isolated receptor biding domain. The results can, for example, help investigate how antibodies bind to the virus. This gives researchers a new tool that may improve understanding of our bodies’ immune response to coronavirus and help to develop medical strategies to overcome COVID-19

SAXS is a powerful technique as it allows researchers to gain insights into protein shape and function at the micro- and nanoscales. The technique has proven to be extremely useful in investigating macromolecular structures such as proteins, especially because it removes the need to crystallize these samples. This means researchers can study the sample in its native form under physiological conditions under which biological reactions occur.

Read more on the European XFEL website

Image: Seen here, the instrument SPB/SFX, where the SAXS experiment was carried out. Using this instrument researchers can study the three-dimensional structures of biological objects. Examples are biological molecules including crystals of macromolecules and macromolecular complexes as well as viruses, organelles, and cells.

Credit: European XFEL / Jan Hosan

Towards a therapy for Parkinson’s disease

Towards a therapy for Parkinson’s disease

Over 100,000 Canadians are living with Parkinson’s disease and 25 more are diagnosed every day, according to Parkinson Canada.

Patients experience tremors, stiffness, and difficulty with movement. Dr. Jean-Francois Trempe, an Associate Professor with McGill University, and colleagues are using the Canadian Light Source (CLS) at the University of Saskatchewan to help search for potential drug targets for the disease.

“I work on a set of proteins that are involved in quality control,” said Trempe. “These proteins are able to sort the damaged proteins from the non-damaged proteins and they send the damaged ones off to be degraded and that’s important for the long-term survival of neurons.”

His team used bright synchrotron light at the CLS to gain insights into a protein involved in formation of flagella, which are important notably for fluid circulation in the brain. By finding new information about this protein, their team is contributing to a body of knowledge that will hopefully lead to a therapy down the road.

Read more and watch the video on the CLS website

The egg in the X-ray beam

The egg in the X-ray beam

Innovative time-resolved method reveals network formation by and dynamics of proteins.

A team of scientists has been using DESY’s X-ray source PETRA III to analyse the structural changes that take place in an egg when you cook it. The work reveals how the proteins in the white of a chicken egg unfold and cross-link with each other to form a solid structure when heated. Their innovative method can be of interest to the food industry as well as to the broad field of research surrounding protein analysis. The cooperation of two groups, headed by Frank Schreiber from the University of Tübingen and Christian Gutt from the University of Siegen, with scientists at DESY and European XFEL, reports the research in two articles in the journal Physical Review Letters.

Eggs are among the most versatile food ingredients. They can take the form of a gel or a foam, they can be comparatively solid and also serve as the basis for emulsions. At about 80 degrees Celsius, egg white becomes solid and opaque. This is because the proteins in the egg white form a network structure. Studying the exact molecular structure of egg white calls for energetic radiation, such as X-rays which is able to penetrate the opaque egg white and has a wavelength that is not longer than the structures being examined.

Read more on the DESY website

Image: When heated, the proteins in the originally transparent chicken egg white form a tightly meshed, opaque network.

Credit: DESY, Gesine Born

Experimental drug targets HIV in a novel way

Experimental drug targets HIV in a novel way

SCIENTIFIC ACHIEVEMENT

Using the Advanced Light Source (ALS), researchers from Gilead Sciences Inc. solved the structure of an experimental HIV drug bound to a novel target: the capsid protein that forms a shield around the viral RNA.

SIGNIFICANCE AND IMPACT

The work could lead to a long-lasting treatment for HIV that overcomes the problem of drug resistance and avoids the need for burdensome daily pill-taking.

Progress in HIV treatment still needed

Over the past couple of decades, safe and effective treatment for HIV infection has turned what was once a death sentence into a chronic disease. Today, people on the latest HIV drugs have near-normal life expectancy.

However, many people are still living with multidrug-resistant HIV, unable to control their virus loads with currently available HIV drugs. The virus develops resistance when people take their pills inconsistently due to forgetfulness, side effects, or a complex schedule. To some, taking a pill every day is a burden: they schedule their lives around it for fear of missing a dose. To others, it is a stigma, as it makes it difficult to hide one’s HIV status from close friends and family.

Read more on the Advanced Light Source website

Image: An experimental small-molecule drug (GS-6207) targets the protein building blocks of the HIV capsid—a conical shell (colored red in this artistic rendering) that encloses and protects the viral RNA—making the virus unable to replicate in cells. Credit Advanced Light Source